Correlating structure, function and dynamics of RNA requires fundamental understanding of structural basis in RNA folding. The objectives of this proposal are to elucidate the structural basis of RNA folding and how ribozymes fold during transcription, a mimic of folding behavior in the cell. The RNA component of bacterial ribonuclease P will be used. Four crystal structures of this ribozyme have been determined in the last three years, enabling us to make fundamental advances in understanding the folding of this large ribozyme. Aim 1 will reveal the major events along the folding pathway of a large RNA by the structural determination of folding intermediates at the residue-level resolution using a combination of experimental data/molecular molecular and cryo-Electron Microscopy. This "first of its kind" look at RNA folding pathways will offer unprecedented insights on the order of structure formation, the interplay between core and peripheral structures, and the role of metal ion-RNA interactions that guide folding and modulate stability. Aim 2 will compare structures of key folding intermediates among two sets of RNAs that have the same core, but distinct peripheral structures. Aim 3 will identify mechanisms for altering folding during transcription, in particular, how a prominent pause site introduced by a specific RNA polymerase affects folding and the assembly of a RNA-protein complex. This broad research program will reveal the structural basis of RNA folding, and the evolutionary and mechanistic link between RNA folding and RNA polymerase.